This invention relates generally to the chemical synthesis of certain peptides and, more particularly, to the use of a mixture of certain of these synthesized peptides as a synthetic vaccine against malaria. This invention also relates to a synthetic hybrid protein copolymer and its use as a vaccine in humans against the asexual blood stages of Plasmodium falciparum induced malaria.
To eradicate malaria, which is dangerously spreading in the developing countries, where more than half of the world's susceptible population lives, scientists are looking for means to control, via chemically synthesized or genetically engineered vaccines, this deadly and threatening parasitic disease.
There are four species of human plasmodia parasites: Plasmodium vivax; P. ovale; P. malariae and P. falciparum. P. falciparum is the most common and lethal. When the malaria infected anopheles mosquito, bites a non-immune person it injects infectious forms of the parasite called sporozoites (first stage) living inside her salivary glands, into the host bloodstream, which within 5 minutes reaches the liver, infecting the liver cells and remains there for about one week.
Each one of these infectious particles or sporozoites divides into 20,000 to 30,000 merozoites (second stage) contained in a bag, or schizont, inside the liver cells. When the cells burst, the merozoites enter the bloodstream and invade circulating red blood cells. Inside these red blood cells (or asexual blood stage), merozoites grow, evolving through several and successive differentiation stages, namely, ring, trophozoite, schizont and merozoite, dividing and breaking out every 48 hours and producing the clinical symptoms of the disease, such as chills and fever, typical of the major forms of malaria. Each new merozoite produces 16-32 offspring that in turn infect new red blood cells, perpertuating the disease and its clinical symptoms.
After several weeks some merozoites differentiate into either male or female gametocytes (third or sexual blood stage) in the bloodstream and when another mosquito bites, it sucks up the malaria infected red blood cells containing gametocytes. Inside the female mosquito the gametocytes break out of the cells and fuse, forming a new generation of sporozoites starting the cycle once again.
A conventional vaccine using attenuated or dead malaria parasites is not feasible due to difficulties in obtaining large amounts of merozoites and contaminating red blood cell debris which potentially will create an autoimmune hemolytic anemia. In fact it was not until 1976 when William Trager developed a laboratory method to enable one to grow small amounts of P. falciparum blood in asexual stages (British Medical Bulletin 38:129, 1982).
The alternatives are then the development by modern techniques, such as chemical synthesis or recombinant DNA, of proteins able to induce protective immunity against the parasite infection.
In this regard, several groups have tried to identify potential targets for immunological attack of the extracellular stages of the parasite, namely, sporozoite, merozoite and gametocytes, the first two being briefly exposed in the blood circulation to the immune system.
Nussenzweig et al. (J. Exp. Med. 156:20, 1982) have identified a protein localized on the surface of sporozoites. Antibodies against this structure confer protection against the experimental disease. It is, however, generally accepted that a vaccine based only on sporozoites will not suffice to prevent malaria. Research to develop a polyvalent vaccine including merozoite and sporozoites targets is actively being pursued in many laboratories throughout the world.
Freeman and Holder (J. Exp. Med. 158:1647, 1983) have characterized a major antigen on the surface of merozoites of 195KD (kilodaltons) protein that generates, upon cleavage, proteins of 83KD, 42KD and 19KD. The first of these, the 83KD protein, remains on the surface of the merozoites. The data suggests that this antigen could be a possible target of protective immunity. Separately, Perrin et al. (J. Exp. Med. 160:441, 1984) have shown protection against blood stage induced malaria in squirrel monkeys when vaccinated with a protein of 140KD.
Perlmann et al. (J. Exp. Med. 159:1686, 1984) have recognized that 155KD protein antigen is invariably deposited on the surface of the P. falciparum infected red blood cells and clinical immunity in endemic areas appears to correlate with antibodies raised against certain parts of this molecule. Collins et al. (Nature 323:259, Sept. 18, 1986) have shown partial protection of Aotus trivirgatus monkeys immunized with genetically engineered fragments of this 155KD protein.
Zavala, et al., Science, 228:1436, 1985 disclose that protective immunity against P. falciparum can be obtained with irradiated sporozoites. The protective antigens, known as circumsporozoite proteins (CS) are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immuno-dominant epitopes formed by tandem repeated sequences of amino acids. It is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide (Asn-Ala-Asn-Pro).sub.3, or (NANP).sub.3.